Journal: Journal of Hematology & Oncology

Article Title: A bispecific CAR-T cell therapy targeting BCMA and CD38 in relapsed or refractory multiple myeloma

doi: 10.1186/s13045-021-01170-7

Figure Lengend Snippet: Preclinical evaluation of BM38 CAR-Ts. a Schematic structure of single-target BCMA CAR, CD38 CAR, bispecific 38BM CAR and BM38 CAR. All the CARs contained a granulocyte–macrophage colony-stimulating factor signal peptide (GM-CSF SP), a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain and a CD3ζ signaling domain. b CAR expression on lentivirus-transduced T cells and CD38 expression on CAR-Ts. Histograms are representative of 3 independent experiments. NT, nontransduced T. NT blank, NT without antibody staining. NT parallel, NT with antibody staining. c Phenotypic profiles of the final CAR-T products and NT cells. Differentiation phenotypes are depicted for naïve (CD45RA + CD62L + ), central memory (CM) (CD45RA − CD62L + ), effector memory (EM) (CD45RA − CD62L − ) and effector (CD45RA + CD62L − ) cells. n = 3, mean ± standard error of mean (SEM). Statistical analysis was performed using one-way ANOVA and subsequent multiple comparisons with BM38 CAR-Ts. *** P < 0.001; ns: not significant. d Expansion curves of the four CAR-Ts and NT cells after lentiviral transduction. n = 5, mean ± SEM. Repeated measures one-way ANOVA was used, ns: not significant. e Cytotoxicity of the four CAR-Ts and NT cells to target cells at an effector: target (E:T) ratio of 1:1, 5:1 and 10:1. n = 4, mean ± SEM. Dunnett’s multiple comparisons test was used at every E:T ratio with BM38 CAR-Ts as the control. * P < 0.05; ** P < 0.01; *** P < 0.001; ns: not significant. f Production of interferon γ (IFNγ) at an E:T ratio of 1:1. n = 3, mean ± SEM. Dunnett’s multiple comparisons test was used with BM38 CAR-Ts as control. ** P < 0.01; *** P < 0.001; ns: not significant. g Luciferase live imaging of MM.1s xenograft mice on day 0, 7, 14 and 23 after infusion of 3.0 × 10 6 NT cells, BCMA CAR-Ts, 38BM CAR-Ts or BM38 CAR-Ts. h Kaplan–Meier survival plot of MM xenograft mice. The log-rank test was used

Article Snippet: All the cell lines were authenticated for CD38 (anti-CD38-APC, 555335; BD Biosciences [BD], USA) and BCMA (anti-BCMA-PE, 130-119-147; Miltenyi Biotec, USA) expression by flow cytometry, and cultured in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (NQBB, China).

Techniques: Expressing, Staining, Transduction, Control, Luciferase, Imaging

Journal: Journal of Hematology & Oncology

Article Title: A bispecific CAR-T cell therapy targeting BCMA and CD38 in relapsed or refractory multiple myeloma

doi: 10.1186/s13045-021-01170-7

Figure Lengend Snippet: Clinical responses mediated of BM38 CAR-Ts. a Swimmer plot for the 23 patients treated in the study. Two patients were enrolled in the two low-dose groups for the consideration of risk–benefit trade-off. b Immunohistochemical staining of patient 13’s right parailiac mass before treatment, showing involvement of MM cells and positive expression of BCMA and CD38. c Computed tomography scans of patient 13 showing a large right parailiac plasmacytoma at enrollment that was partially eliminated in month 3 and completely disappeared in month 5. d Changes of serum M protein in patient 13 during BM38 CAR-T treatment. e BCMA and CD38 expression on MM cells in patient 15 and 20 at baseline. Representative staining and gating of MM cells are shown in Additional file : Fig. S6

Article Snippet: All the cell lines were authenticated for CD38 (anti-CD38-APC, 555335; BD Biosciences [BD], USA) and BCMA (anti-BCMA-PE, 130-119-147; Miltenyi Biotec, USA) expression by flow cytometry, and cultured in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (NQBB, China).

Techniques: Immunohistochemical staining, Staining, Expressing, Computed Tomography

Journal: Journal of Hematology & Oncology

Article Title: A bispecific CAR-T cell therapy targeting BCMA and CD38 in relapsed or refractory multiple myeloma

doi: 10.1186/s13045-021-01170-7

Figure Lengend Snippet: Predictive docking patterns of BM38 CAR to MM cells. a Hypothetical structure of the anti-BCMA scFv and anti-CD38 scFv joined with an (EAAAK) 3 linker (not shown). b Most favorable docking models of the anti-BCMA scFv with 51 amino acid residues of the extracellular domain of BCMA (2kn1.pdb); c Most favorable docking models of the anti-CD38 scFv with 257 amino acid residues of the extracellular domain of CD38 (1yh3.pdb); d Dual docking of BM38 CAR with BCMA and CD38. e Theoretical single combination pattern of the anti-CD38 scFv and CD38 on MM cells. f Theoretical single combination pattern of the anti-BCMA scFv and BCMA on MM cells. g Theoretical double-binding pattern of the bispecific BM38 CAR to BCMA and CD38 on MM cells. h–i The CD38 extracellular domain contains 257 amino acids and the BCMA extracellular chain consists of only 54 amino acids. The anti-BCMA scFv and anti-CD38 scFv contain 246 and 249 amino acids, respectively. Theoretically, CD38 binding might assist the anti-BCMA scFv in binding to BCMA on myeloma cells, and BCMA coalescence would reversely strengthen CD38 binding

Article Snippet: All the cell lines were authenticated for CD38 (anti-CD38-APC, 555335; BD Biosciences [BD], USA) and BCMA (anti-BCMA-PE, 130-119-147; Miltenyi Biotec, USA) expression by flow cytometry, and cultured in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (NQBB, China).

Techniques: Binding Assay

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

Article Title: Blockade of BAFF Receptor BR3 on T cells Enhances Their Activation and Cytotoxicity

doi: 10.1097/CJI.0000000000000209

Figure Lengend Snippet: (A) Flow cytometric analysis of CD25 expression. Purified CD4+ and CD8+ T cells were incubated with goat polyclonal BR3, TACI, or BCMA specific neutralization antibodies or the goat IgG control and activated with plate-bound CD3/CD28 specific stimulatory antibodies for 24 hours. Anti-CD25 APC was used to detect CD25 expression which is gauged by percent positive CD25 cells. Five separate T cell donors were analyzed. (B) Flow cytometric analysis as for (A) measured by mean channel fluorescence of CD25. (C) Relative gene expression of CD25 using semi-quantitative PCR. Purified T cells from three healthy donors were incubated with either the anti-BR3 neutralization antibody or the goat IgG control and activated for 24 hours.

Article Snippet: Antibodies used were as follows: anti-BR3 PE, clone 11C1, BD Biosciences; anti-TACI APC, clone 165004, and anti-BCMA APC, goat pAb FAB193A, both from RnD Systems; anti-CD25 FITC-Violet and APC, clone 3H3, Miltenyi Biotec; anti-CD69 FITC, clone FN50, eBioscience; anti-IFN-γ APC, clone B27, BD Biosciences; anti-Granzyme B PE, clone GB11, BD Biosciences; anti-CRTAM PE, clone Cr24.1, Biolegend, Inc; anti-BAFF APC, clone 1D6, BD Biosciences; anti-BCMA APC, pAb FAB193A, RnD Systems, Inc; anti-cleaved PARP (Asp214) PE, clone XF21-852, BD Biosciences; anti-active Caspase 3 FITC, clone C92-605, BD Biosciences.

Techniques: Expressing, Purification, Incubation, Neutralization, Fluorescence, Real-time Polymerase Chain Reaction